Subunit Structure of Qβ Replicase

Poliovirus-infected HeLa cells were labeled with radioactive methionine or phenylalanine and subjected to a new purification procedure for the viral induced RNA polymerase activity. Detergent-solubilized polymerase activity was purified by precipitation with 2 M LiCl and sedimentation through sucrose gradients. Approximately 0.901% of the incorporated amino acid radioactivity sediments with the peak of polymerase activity. Gradient fractions comprising the polymerase activity peak were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and found to contain predominantly one virus-specific polypeptide. Polyacrylamide gel electrophoresis also reveals that this purified polypeptide migrates with a 58,000 molecular weight noncapsid poliovirus polypeptide.At least 14 virus-specific polypeptides can be detected in poliovirus-infected HeLa cells (1), all derived from post-translational proteolytic cleavage of a large precursor molecule (2-4). Although the general relationship of these polypeptides to the cleavage pattern is known (5), no enzymatic function has been assigned to any of the viral polypeptides. Poliovirus RNA polymerase activity is easily measured in crude extracts (6, 7), but the lack of a suitable purification procedure has prevented the identification of the viral polypeptides necessary for the enzymatic activity. Previous attempts to purify the polymerase activity from picornavirus-infected cells have shown that the polymerase activity sediments as a membraneassociated complex of 60-250 S (8-14) and is associated with many viral polypeptides (15).We have found that precipitation with 2 M LiCl results in significant purification of the polio polymerase complex. Details of the purification procedure and properties of the purified polymerase will be reported elsewhere (Lundquist and Maizel, manuscript in preparatron). Here cell of purified Mahoney strain poliovirus type 1. Actinomycin D (4 ,Ag/ml) was added 10 min after infection. The radioactive amino acid (25 ,Ci/ml) was added to infected cells incubated in medium containing normal levels of all other essential amino acids. The medium concentration of the radioactive amino acid was reduced to 2% of the concentration normally present in Eagle's minimum essential medium. The medium was supplemented with 5% fetal calf serum 30 min after infection. Levels of guanidine that have no discernible effect on HeLa cells inhibit the replication of poliovirus, but viral inhibition of host cell protein synthesis is unaffected (1). Virus-specific proteins can then be labeled after reversal of guanidine inhibition. Guanidine (1 mM) was added 15 min after infection with poliovirus. After incubation at 370 for 1.5 hr, the infected cells were harvested by centrifugation, washed with Earle's isotonic salt solution, and resuspended in medium without guanidine, containing 5% fetal calf serum. The radioactive amino acid was added after 15 min, and the infection was stopped 2.5 hr after removal of guanidine.The mechanism of guanidine inhibition is u...

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“…Q,3 replicase has been extensively purified and exists as a complex of three host polypeptides and one viral polypeptide (26,27 …”

Section: Discussionmentioning

confidence: 99%

Isolation of a Viral Polypeptide Associated with Poliovirus RNA Polymerase

Lundquist

Ehrenfeld

Maizel

1974

Proc. Natl. Acad. Sci. U.S.A.

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“…Qb replicase consists of four different subunits, one of which (b) is encoded by the phage genomic RNA 5,6 and the other three are host (Escherichia coli) proteins involved in protein synthesis: ribosomal protein S1 [7][8][9] and elongation factors EF-Tu and EFTs 10 . While the roles of the elongation factors remain unclear, the function of S1 in Qb replicase seemed to be well established as long as 40 years ago, when it was for the first time separated from the three-subunit core enzyme 7 .…”

mentioning

confidence: 99%

Ribosomal protein S1 functions as a termination factor in RNA synthesis by Qβ phage replicase

et al. 2013

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S1 is the largest ribosomal protein, and is vitally important for the cell. S1 is also a subunit of Qb replicase, the RNA-directed RNA polymerase of bacteriophage Qb. In both protein and RNA syntheses, S1 is commonly believed to bind to a template RNA at the initiation step, and not to be involved in later events. Here, we show that in Qb replicase-mediated RNA synthesis, S1 functions at the termination step by promoting release of the product strand in a single-stranded form. This function is fulfilled by the N-terminal fragment comprising the first two S1 domains. The results suggest that S1 might also have a role other than mRNA binding in the ribosome.

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“…Until now, phage RNA replication has been mainly studied in Qfl. The core of Qfl RNA polymerase contains one phage-specified protein and three bacterial proteins (Kamen, 1970;Kondo et al, 1970;Blumenthal et aL, 1972;Inouye et al, 1974). An additional protein, called host factor (HF) has been implicated in Qfl RNA replication in vitro (Shapiro et al, 1968;Carmichael et al, 1975).…”

Section: Discussionmentioning

confidence: 99%